Project Summary/Abstract Phosphorylation of histidine (His) in proteins has a 60-year history, initially identified as a P-enzyme intermediate, but subsequently as a regulatory mechanism in bacteria essential for signal transduction by surface receptors that sense nutrients. Such signaling systems are lacking in mammals, but phosphohistidine (pHis) is not only a key P-enzyme intermediate in mammalian enzymes (e.g. NME1/2, ACLY), but also occurs as a reversible end-state protein modification, e.g. pHis18 in histone H4. His phosphorylation is labile to acid and heat making it challenging to study, and to circumvent this our group developed a series of monoclonal antibodies (mAbs) that recognize the 1-pHis or 3-pHis isoforms in a sequence-independent manner. These mAbs were used to detect pHis in cells by immunoblotting and immunofluorescence (IF) staining, and for affinity enrichment of pHis proteins for MS analysis, revealing ~700 potential pHis proteins and implying the existence of large ?hidden? phosphoproteome not detectable by conventional methods. In a collaborative study, these mAbs were used to demonstrate site and isoform specific His phosphorylation of the KCa3.1 K+ channel and deduce how pHis triggers channel opening. In a second collaborative study, the mAbs were used to demonstrate increased levels of pHis proteins in mouse and human hepatocellular carcinoma (HCC), and show that the increase was due to reduced expression of the LHPP pHis phosphatase in the tumors, suggesting that LHPP acts as a tumor suppressor, and that elevated His phosphorylation plays a driver role in this cancer. On this basis, studies are planned to investigate whether increased His phosphorylation plays a broader role in human cancer. Initially, our structures of mAb-derived Fab fragments bound to pHis peptides will be exploited to develop better tools for studying His phosphorylation in cancer - mAbs with higher affinity and scFvs for intracellular expression to localize and perturb pHis proteins, sequence specific pHis antibodies for studying individual proteins, and improved MS-based pHis site identification. In parallel, in-depth studies with existing 1/3-pHis mAbs, as well as new pHis reagents as they come online, will be conducted on three selected tumor types - HCC, pediatric neuroblastoma and pancreatic cancer, where there is evidence that aberrant His phosphorylation may play a role. Immunoblotting, and IF and IHC staining will be performed on tumor tissues/cell lines and normal controls, combined with use of optimized pHis peptide enrichment and site identification protocols to define changes in His phosphorylation unique to tumor tissues. Where warranted, the function of individual pHis sites in cancer will be studied by site-directed mutagenesis in tumor cell lines. In each case, further experiments will be guided by the identity and function of pHis proteins found in a particular cancer. Overall, it is anticipated that comparative studies on HCC, neuroblastoma and PDAC will shed light on whether His phosphorylation plays a general role in cancers, whether there are common mechanisms, and whether targeting His phosphorylation could be a viable new therapeutic approach.